Recombinant therapeutic proteins have had a significant impact on the clinical treatment of diseases, including cancer, over the past few decades. There are over 630 recombinant proteins and peptides in commercial development, and protein-derived therapeutics continue to grow rapidly relative to small molecule therapeutics. As more recombinant proteins enter the pharmaceutical market, the potential risks associated with these products are becoming more of a concern. In particular, therapeutic proteins, unlike small molecules, may be unstable and prone to aggregation (Chi et al., Physical stability of proteins in aqueous solution: Mechanism and driving forces in nonnative protein aggregation, Pharmaceutical Research 20(9):1325-1336 (2003). Protein aggregation can compromise the safety and effectiveness of the product.
Even though industry and regulatory agencies are aware of aggregation and have policies and guidelines for their detection in therapeutic protein compositions, some aggregates still go undetected, in-part due to limitations of the conventionally accepted analytical techniques. For instance, the USP currently has no guidelines for detection of particles 0.1 to 10 microns in size. Protein aggregates less than 0.1 micron are detected by analytical methods such as size exclusion chromatography, and particles greater than 10 microns are detected by the USP light obscuration <788> technique. There are no clear recommendations for detection of particles greater than about 0.1 micron but less than about 10 microns, and the significance of these particles to the immunogenic potential of the product has not been demonstrated. This gap in subvisible particle detection leaves an opportunity for protein aggregates to exist in approved commercial products and current biologics undergoing development.
Protein aggregation occurs due to colloidal or conformation instability allowing proteins to assemble with concomitant loss of native structure and activity. Stresses such as freeze-thawing, agitation (e.g. air-water interface), and UV light exposure, are commonly encountered during processing, shipping, and storage of a therapeutic product and are known to aggregate proteins (Chi et al. 2003). Aggregates may also be generated during protein purification as the protein moves through a variety of solution exchanges at high protein concentrations on column surfaces. Protein aggregation may proceed through specific pathways that are initiated by instability of the native protein conformation or colloid instability associated with protein-protein interactions. Conditions such as temperature, solution pH, ligands and cosolutes, salt type and concentration, preservatives, and surfactants all modulate protein structure and protein-protein interactions, and thus aggregation propensity.
Aggregates produced as a result of different stresses may exhibit different size distributions and their component proteins may contain different secondary and tertiary structures, which presumably expose different epitopes, potentially provoking immune responses (Seefeldt et al., High-pressure studies of aggregation of recombinant human interleukin-1 receptor antagonist: thermodynamics, kinetics, and application to accelerated formulation studies, Protein Sci. 14(9):2258-66 (2005)).
Protein aggregates present in therapeutic protein compositions may not be recognized as “natural” by the immune system. This might be due to exposure of a new epitope in the aggregated protein that is not exposed in the non-aggregated protein, or by formation in the aggregate of a new epitope, with the result that the immune system is sensitized to the administered recombinant protein aggregate. While in some instances the immune system produces antibodies to the aggregates that do not neutralize the therapeutic effect of the protein, in other cases, antibodies are produced that bind to the recombinant protein and interfere with the therapeutic activity, thereby resulting in declining efficacy of the therapy.
Repeated administration of a recombinant protein can cause acute and chronic immunologic reactions (Schellekens, H., Nephrol. Dial. Transplant. 18:1257 (2003); Schellekens, H., Nephrol. Dial. Transplant. 20 [Suppl 6]:vi3-vi9 (2005); Purohit et al. J. Pharm. Sci. 95:358 (2006)). This loss or “breaking” of tolerance can have serious effects including the development of autoimmune diseases. For example, upon repeated administration of a recombinant protein, tolerance can be broken, and an immune response produced against the recombinant protein may cross-react with the individual's endogenous protein. A mechanism for breaking self-tolerance was demonstrated in transgenic mice immune tolerant for human interferon-alpha 2. When preparations containing aggregates of recombinant human interferon-alpha 2b were administered to the mice, the mice lost tolerance for interferon-alpha 2 in a dose-dependent manner (see Hermeling et al., J Pharm Sci. 95:1084 (2006)).
A loss of tolerance to an endogenously produced protein was observed in patients using a preparation of recombinant erythropoietin. Certain preparations of erythropoietin sold under the trademark EPREX (Johnson & Johnson, New Brunswick, N.J.) in Europe were found to break the immune tolerance of patients for their own endogenous erythropoietin, leading to antibody-mediated pure red cell aplasia (PRCA). The exogenous erythropoietin preparation, which was administered to correct a deficiency in red blood cell production, elicited the patient's immune system to produce antibodies that neutralized endogenously produced erythropoietin, causing a complete block in differentiation of red blood cells. The cause of the immune response has been attributed to leachates in the preparation which formed adjuvants with erythropoietin (Boven et al., Nephrol. Dial. Transplant. 20 Suppl 3:iii33 (2005)), although other factors, such as aggregates, may also be involved (Schellekens and Jiskoot, Nature Biotech. 24:613 (2006)).
Accordingly, new protein engineering and manufacturing strategies are needed to minimize immunogenicity of protein therapeutics and improve the effectiveness of therapy.